Medicinal compositions for the improvement of blood coagulation comprising TCF-II

ABSTRACT

Blood coagulation normalizing agents comprising effective ingredient of TCF-II. 
     The blood coagulation normalizing agents of the present invention comprised of glycoprotein TCF-II produced by human fibroblast cells and accelerates the production of blood coagulation factor and increases platelet count to treat abnormal blood coagulation such as disseminated intravascular coagulation (DIC) and decreased platelet count caused by administration of cisplatin.

FIELD OF THE INVENTION

This invention relates to medicinal compositions for the normalizationof blood coagulation or treatment of abnormal blood coagulation,comprising a glycoprotein produced by human fibroblast cells, TCF-II(tumor cytotoxic factor-II), more particularly agents for the treatmentof disseminated intravascular coagulation (DIC) syndrome, increase ofplatelet count and prevention of thrombocytopenia.

DESCRIPTION OF THE PRIOR ART

Biologically active substances produced by human derived fibroblastcells, for example, β-interferon as a tumor cytotoxic factor, have beenwell known. Biologically active substances produced by fibroblast cellsother than β-interferon such as a tumor cytotoxic glycoprotein calledCBF is disclosed in Japanese Patent Laid-open Publication No. 146293(1983). A tumor growth inhibitory factor (INF) with molecular weight of35,000-48,000, which is purified from culture broth of fibroblastsderived from human tissue, is disclosed in Japanese Patent Laid-openPublication No. 33120 (1986). Furthermore, a tumor necrosis factor-likesubstance which is purified from the culture broth of fibroblasts, afibroblast-derived necrosis factor, FNF, and a physiologically activesubstance with cytotoxic activity, which is produced by animal-derivedfibroblasts and has a molecular weight of 40,000 to 60,000 and anisoelectric point of pH 5.0±0.5, are disclosed in Japanese PatentLaid-open Publication No. 1872 (1986) and Japanese Patent Laid-open No.103021 (1987), respectively. In addition, all the primary amino acidsequence and cDNA encoding the amino acid sequence of a tumor cytotoxicfactor, which is obtained from the culture broth of human-derivedfibroblasts, with a molecular weight of 36,000±1,000 and an isoelectricpoint of pH 10.5 or higher are disclosed in Japanese Patent Laid-openNo. 10988 (1989). The inventors have been investigating antitumorsubstances derived from human fibroblast cells and found a new antitumorprotein, which is different from antitumor proteins hitherto reported.Furthermore, the inventors successfully cloned a cDNA encoding theprotein, determined its all the primary amino acid sequence, andconfirmed usefulness of the protein. The new antitumor protein and itsgene were disclosed in the inventors' International Patent PublicationNo. 10651 (1990). The new antitumor protein was called as TCF-II.

TCF-II has both potent antitumor activity and proliferation stimulativeactivity for normal cells and is a member of the HGF (hepatocyte growthfactor) group. Molecular weight determination of TCF-II with SDSelectrophoresis showed a common band, A chain, with 78,000±2,000 and twobands, B and C chains, with 30,000±2,000 and 26,000±2,000, respectively.

TCF-II may be used for the regeneration of liver after the hepatectomydue to its proliferation stimulative effect on hepatocytes. However, noeffect of TCF-II on diseases related to abnormal blood coagulation suchas DIC and thrombocytopenia has been known.

DIC accompanies with various basal diseases and frequently causes bloodclotting in systemic fine blood vessels. It impairs functions of manyorgans and tends to result in marked hemorrhages due to consumptioncoagulopathy and hyperfibrinolysis. The basal diseases causing DICinclude malignant tumor, leukemia, sepsis, infectious diseases,obstetric diseases such as placental abruption and amniotic fluidembolism, and shock in a descending order. Diseases frequentlyaccompanied with DIC include leukemia, malignant lymphoma, myeloma,collagen disease, and liver diseases in. descending order. In additionto risk factors such as surgical invasion, administration of anantitumor agent, infectious diseases and shock may accelerate the onsetof DIC. During the onset of DIC, coagulation factors such as bloodcoagulation factors I, II, V, VIII and XIII are consumed and their bloodlevel decrease. Platelets are consumed in thrombus of systemic fineblood vessels by adhesion and coagulation in damaged subcutaneoustissues and incorporation in fibrin. Fibrinolytic system is activated byinflux of tissue activator into blood stream, formation of kallikreinand so forth after the activation of coagulation system and the fibrindegradation products (FDP) are formed by secondary fibrinolysis.Decrease of plasma fibrinogen, elongation of prothrombin time anddecrease of platelet count can markedly be observed. Widely spreadtendency of hemorrhage has been known to be due to massive consumptionof antithrombin III or α₂ -antiplasmin caused by inactivation ofthrombin and plasmin. Furthermore, DIC is known to exhibit symptoms ofdisturbed production of vitamin K dependent coagulation factor due toliver damage, intoxication and other causes, abnormal protein metabolismincluding cachexia and adverse effects of medicines, bleeding andinsufficient hemostasis due to thrombocytopenia.

Heretofore, no effective treatment of DIC has been found and accordingto symptoms of DIC, only palliative therapy such as administration ofwarfarin and fibrinogen to hemorrhagic tendency have been performed. Inaddition, thrombocytopenia also occurs by the administration ofmedicines including antitumor agents and by various diseases, but nopreventive treatment to the decrease of platelets has been found.

DISCLOSURE OF THE INVENTION

The inventors noticed the biological activity of TCF-II and have beeninvestigating the use of TCF-II as an antitumor agent and a diagnosticmarker of the diseases.

The inventors found in the investigation on actions of TCF-II for liverthat TCF-II provides not only proliferation of hepatocytes but alsotherapeutic effects on various abnormal blood coagulation accompanied byDIC. Furthermore, the inventors found that TCF-II dose dependentlyincreases platelet count and inhibits the decrease of platelet count dueto administration of antitumor agents. Heretofore, the therapeuticeffect of TCF-II on various blood coagulation disorder such asdisseminated intravascular coagulation (DIC) or prevention againstdecrease of platelet count has not been confirmed. The increase ofplatelet count and therapeutic effect of TCF-II on abnormal bloodcoagulation and thrombocytopenia are surprising.

An object of the invention is to provide a treatment agent comprisingTCF-II as an effective ingredient against abnormal blood coagulation.

The agents containing TCF-II as an effective ingredient to normalizeblood coagulation practically include blood coagulation normalizingagent, platelet increasing agent and thrombocytopenia preventive agent.

The effective ingredient of the present invention is a knownglycoprotein (TCF-II) derived from human fibroblast cells as describedpreviously.

On SDS-polyacrylamide gel electrophoresis, TCF-II showed bands with M.W.78,000±2,000 and 74,000±2,000 under nonreduced conditions, and alsoshowed a common band A with M.W. 52,000±2,000 in the reduced conditionand band B with M.W. 30,000±2,000 and band C with M.W. 26,000±2,000under reduced conditions. TCF-II also showed an isoelectric point of pH7.4-8.6 and was determined as a glycoprotein having the primary aminoacid sequence composed of 723 amino acids.

The above mentioned TCF-II can be obtained by concentration of a humanfibroblast cell culture broth, adsorption and solution by ion exchangechromatography of the concentrate and affinity chromatography of theeluate (WO90/10651) or by a genetic engineering method (WO92/01053).

TCF-II obtained from human fibroblast cells cultured by the methoddisclosed in WO90/10651 can be used. Furthermore, TCF-II produced by agene engineering technique using microorganisms or other cellstransfected with the gene having oligonucleotide sequence disclosed inthe above mentioned patent publication may be used. The production ofTCF-II by the genetic engineering method may be carried out by themethod invented by the present inventors and disclosed in WO92/01053. Inaddition, TCF-II analogues having different sugar chains or no sugarmoieties produced by different host cells or microorganisms may also beused. However, presence of sugar moieties is preferable because of theirparticipation in the in vivo metabolic rate.

TCF-II can be concentrated and purified by conventional isolation andpurification methods, for example, precipitation with an organicsolvent, salting-out, gel filtration chromatography, affinitychromatography using a monoclonal antibody and electrophoresis. Thepurification by affinity chromatography using a monoclonal antibodydisclosed in Japanese Patent-Application No. 177236 (1991) by thepresent inventors may be applied.

The resultant purified TCF-II may be kept under lyophilization or deepfreezing.

Furthermore, the blood coagulation normalizing agent of the presentinvention may be administered as injection preparations intravenous, andany route such as intraarterial, intramuscular and subcutaneousinjections can be selected. Blood coagulants such as fibrinogen,coagulation controlling factors such as antithrombin III and drugs suchas FOY, a protease inhibitor used for the treatment of DIC are also usedconcomitantly.

The injection preparations of TCF-II may be used alone or in combinationwith above mentioned medicines and adjuvants such as human serumalbumin, surface active agents, amino acids and sugars.

The doses of TCF-II included in the blood coagulation normalizing agentsof the present invention can be determined according to the symptoms andconditions and the age of the patients and generally administered in adose range of 100-30,000 μg TCF-II/kg, preferably, 500-3,000 μgTCF-II/kg, 1-7 times a week, Long term administration may be usedaccording to the symptoms and conditions of the patients.

Following diseases may be illustrated as indications of the presentinvention.

(1) Various diseases accompanying decrease of platelet, particularlydecreased production of platelet due to radiation damage and medicinessuch as antitumor agents, aplastic anemia and related diseases,substitution of hematopoietic tissue due to myelophthisis, leukemia,malignant lymphoma, myelofibrosis and metastasis of tumors to bone,megalocytic anemia due to deficiency of vitamin B₁₂ and folic acid,production of ineffective platelet due to paroxysmal nocturnalhemoglobinuria, congenital low production of platelet and thrombopoietindeficient infections such as sepsis and AIDS.

(2) Various diseases accompanied with accelerated platelet destruction,particularly DIC, disturbance of fine blood vessels, massive bloodtransfusion, hyperglyceridemia, drug induced destruction, thromboticthrombocytopenic purpura (TTP), extracorporal circulation such as renaldialysis, hemangioma, destruction by immune complex such as idiopathicthrombocytopenic purpura, Evans syndrome and drug allergy.

(3) Abnormal distribution of platelet, particularly retention ofplatelets and decreased production of platelets accompanied withsplenoma and splenic hyperfunction, and bone marrow fibrosis accompaniedby extramedullarly hematopoiesis.

(4) Diseases accompanying abnormal platelet functions such as abnormalplatelet adhesion, abnormal platelet, and abnormal platelet coagulation,particularly thrombasthenia, afibrinogenemia, dysproteinemia such asmyeloma, acute myelocytic leukemia, diabetes, myeloproliferativedisorder, homocystinuria, storage pool disease, Bernard-Souliersyndrome, von Willebrand's disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the increase of platelet count by administration of theagent of the present invention in healthy rats.

FIG. 2 shows the shortening effect of prothrombin time by administrationof the agent of the present invention in healthy rats.

FIG. 3 shows the shortening effect of prothrombin time in bloodcoagulation disturbed rats by 70% hepatectomy followed by administrationof the treatment agent of the present invention.

FIG. 4 shows the increase of plasma fibrinogen in blood coagulationdisturbed rats by 70% hepatectomy followed by administration of thetreatment agent of the present invention.

FIG. 5 shows the shortening of thrombotest value in blood coagulationdisturbed rats by 70% hepatectomy followed by administration of thetreatment agent of the present invention.

FIG. 6 shows the changes of antithrombin III activity in antithrombinIII activity decreased rats by administration of the treatment agent ofthe present invention.

FIG. 7 shows the changes of thrombotest value in antithrombin IIIactivity decreased rats by administration of the treatment agent of thepresent invention.

FIG. 8 shows the changes of prothrombin time in DIC rats byadministration of the treatment agent of the present invention.

FIG. 9 shows the changes of plasma fibrinogen concentration in DIC ratsby administration of the treatment agent of the present invention.

FIG. 10 shows the changes of antithrombin III activity in DIC rats byadministration of the treatment agent of the present invention.

FIG. 11 shows the changes of thrombotest value in DIC rats byadministration of the treatment agent of the present invention.

FIG. 12 shows the changes of prothrombin time in DIC rats byadministration of the treatment agent of the present invention.

In the FIGS., * indicates P<0.05 and ** indicates P<0.01.

THE BEST MODE FOR CARRYING OUT THE PRESENT INVENTION

The present invention will be explained in more detail by the followingexamples.

Example 1

Purification of TCF-II

Purified TCF-II was obtained by cell culture according to the methoddisclosed in WO90/10651 or disclosed by Higashio, K. et al. (B.B.R.C.,170, 397-404, 1990).

Human fibroblast cells, IMR-90 (ATCC CCL 186), 3×10⁶ cells, wereinoculated in I00 ml of DMEM medium containing 5% calf serum in a rollerbottle and cultured by rotating at a rate of 0.5-2/min. for seven days.The culture was continued up to total cell numbers of 1×10⁷ cells intotal, the proliferated cells were trypsinited and collected at thebottom of the bottle. In the bottle, 100 g of sterilized 5-9 meshceramic (Toshiba Ceramic Co., Ltd.) was placed and cultured for 24 hrs.upon standing. Then, 500 ml of the culture medium shown above was addedto the bottle and cultured further. The total culture medium wasrecovered every 7-10 days and fresh culture medium was supplied forfurther culture. Thus, the culture was continued for two months and theculture broth of four l/bottle was recovered.

The combined culture solution showed specific activity of 32 U/ml.

Ultrafiltration of 750 l of the cultured solution was performed using amembrane filter (Amicon Corp., MW 6,000 cut) and TCF-II in theconcentrate was purified by chromatographies consisting of five stepsusing CM Sephadex C-50 (Pharmacia Biosystems Corp.), ConA Sepharose(Pharmacia Biosystems Corp.), MonoS column (Pharmacia Biosystems Corp.)and heparin Sepharose (Pharmacia Biosystems Corp.). The purified TCF-IIhas specific activity of 5,248,000 U/mg.

Example 2

Production of recombinant TCF-II

TCF-II gene recombinant cells were cultured according to the methoddisclosed in WO92/01053 and purified TCF-II was obtained. TransformedNamalwa cells were cultured and 20 l of the culture solution wasobtained. The culture solution was treated successively bychromatographies using CM-Sephadex C-50 column, Con-A Sepharose CL-6Bcolumn and MonoS column to give approximately 11 mg of active TCF-II.

Example 3

Production of pharmaceutical compositions of TCF-II

In the present examples, recombinant TCF-II obtained by Example 2 wasused for the production of injection preparations.

(1) TCF-II 20 μg

Human serum albumin 100 mg

This composition was dissolved in 0.01M PBS at pH 7.0 and adjusted to 20ml in total. The solution was sterilized, divided into vials (2 mleach), lyophilized and sealed.

    ______________________________________                                        (2)      TCF-II            40     μg                                                Tween 80          1      mg                                                   Human serum albumin                                                                             100    mg                                          ______________________________________                                    

This composition was dissolved in a saline solution for injection andadjusted to 20 ml in total. The solution was sterilized, divided intovials (2 ml each), lyophilized and sealed.

    ______________________________________                                               (3) TCF-II         20    μg                                                    Tween 80       2     mg                                                       Sorbitol       4     g                                             ______________________________________                                    

This composition was dissolved in 0.01M PBS at pH 7.0 and adjusted to 20ml in total. The solution was sterilized, divided into vials (2 mleach), lyophilized and sealed.

    ______________________________________                                               (4) TCF-II         40    μg                                                    Tween 80       2     mg                                                       Glycine        2     g                                             ______________________________________                                    

This composition was dissolved in a saline solution for injection andadjusted to 20 ml in total. The solution was sterilized, divided intovials (2 ml each), lyophilized and sealed.

    ______________________________________                                               (5) TCF-II         40    μg                                                    Tween 80       1     mg                                                       Sorbitol       2     g                                                        Glycine        1     g                                             ______________________________________                                    

This composition was dissolved in a saline solution for injection andadjusted to 20 ml in total. The solution was sterilized, divided intovials (2 ml each), lyophilized and sealed.

    ______________________________________                                        (6)      TCF-II             20    μg                                                Sorbitol           4     g                                                    Human serum albumin                                                                              50    mg                                          ______________________________________                                    

This composition was dissolved in 0.01M PBS at pH 7.0 and adjusted to 20ml in total. The solution was sterilized, divided into vials (2 mleach), lyophilized and sealed.

    ______________________________________                                        (7)      TCF-II             40    μg                                                Glycine            2     g                                                    Human serum albumin                                                                              50    mg                                          ______________________________________                                    

This composition was dissolved in a saline solution for injection andadjusted to 20 ml in total. The solution was sterilized, divided intovials (2 ml each), lyophilized and sealed.

(8) TCF- H 10 mg

Human serum albumin 100 mg

This composition was dissolved in 0.01M PBS at pH 7.0 and adjusted to 20ml in total. The solution was sterilized, divided into vials (2 mleach), lyophilized and sealed.

Above mentioned TCF-II pharmaceutical preparations shown by (1) to (8)can be used for the treatment of abnormal blood coagulation andprevention of decrease of platelet count caused by administration ofantitumor agents according to the above mentioned dosage.

Treatment agents to abnormal blood coagulation containing TCF-II as aneffective ingredient are provided by the present invention. Hereinafter,test experiments with the treatment agents prepared according to thepresent invention will be shown to confirm the therapeutic effects andexplain the present invention.

Experiment 1

Effects on the increase of platelet count and on prothrombin time

(1) Method

Male Wistar rats, seven week old, six in one group, were used for theexperiments. TCF-II was intravenously administered every 12 hrs., 28times in total repeatedly at doses of 0 (zero), 5, 50, 500, and 5,000μg/kg, respectively, and blood was drawn at 12 hrs. after the finaladministration to determine platelet count and prothrombin time.

The injection preparation of TCF-II was prepared by dissolving TCF-II in10 mM phosphate buffer, pH 6.8-7.2, containing 0.01% Tween 80, 0.25%human serum albumin and 0.15M NaCl. The preparation was used in all thefollowing experiments.

(2) Results

Increase of platelet count (FIG. 1) and shortening of prothrombin time(FIG. 2) were dose dependently observed. Both parameters showed markedimprovement at doses of 500 μ/kg or over. The therapeutic effect onabnormal blood coagulation was confirmed.

Experiment 2

Effect on prothrombin time, plasma fibrinogen level and thrombotestvalue in blood coagulation disturbed rats by hepatectomy

Coagulating factors are produced in the liver, thus rats with decreasedcoagulation ability were prepared by the hepatectomy and the effect ofTCF-II was confirmed using these model animals.

(1) Method

In seven week old male Wistar rats, six in one group, 70% of the liverwas resected and TCF-II was intravenously administered repeatedly tothese rats at doses of 0 (zero), 20, 100 and 500 μg/kg, respectively,every 12 hrs. for two days, four times in total. The blood was drawn at48 hrs. after the start of the administration and prothrombin time,plasma fibrinogen level and thrombotest value were determined.

(2) Results

Shortening of prothrombin time (FIG. 3), increase of plasma fibrinogen(FIG. 4) and shortening of thrombotest value (FIG. 5) were observed withthe administration of TCF-II. The therapeutic effect of the presentinvention on the liver oriented disease of blood coagulation isconfirmed.

Experiment 3

Therapeutic effect of TCF-II administration rat model with decreasedantithrombin III

(1) Method

To seven week old male Wistar rats, 10 in one group, 250 mg/kg/day ofDL-ethionine was administered for four days to prepare antithrombin IIIactivity decreased model. TCF-II was administered to the model at dosesof 0 (zero), 50 and 500 μg/kg, respectively, every 12 hrs. intervals.Prothrombin time, antithrombin III activity and thrombotest value weredetermined at 48 hrs. after the start of the administration.

(2) Results

Recovery of antithrombin III activity (FIG. 6), shortenings ofthrombotest value (FIG. 7) and prothrombin time (FIG. 8) were observedwith the administration. The treatment agent of the present inventionincreased antithrombin III level confirming the efficacy for thetreatment of blood coagulation disease.

Experiment 4

Therapeutic effect on DIC model

(1) Method

To seven week old male Wistar rats, 10 in one group, 500 mg/kg ofgalactosamine was subcutaneously administered immediately after 70%hepatectomy to prepare a pathologic model of liver damage and DICsymptoms. TCF-II was intravenously administered repeatedly at a dose of500 μg/kg every 12 hrs. for two days and four times in total. Blood wasdrawn at 48 hrs. after the start of the administration and prothrombintime, plasma fibrinogen level, antithrombin III level and thrombotestvalue were determined.

(2) Results

Increases of plasma fibrinogen level (FIG. 9) and antithrombin IIIactivity (FIG. 10), and shortenings of thrombotest value (FIG. 11) andprothrombin time (FIG. 12) were observed with the administration. Thus,the efficacy of the present invention on disturbed blood coagulation dueto DIC was confirmed.

As shown above, the therapeutic effect of the present invention to bloodcoagulation disease was confirmed.

Experiment 5

Therapeutic effect on platelet decreased model due to administration ofantitumor agent

(1) Method

Seven week old male Wistar rats, 10 in one group, were administered onceeight mg/kg of cisplatin (Shionogi & Co., Ltd.) to prepare plateletcount decreased model.

Then, TCF-III was intravenously administered at a dose of one mg/kgimmediately after the administration of cisplatin and at 12 hrs.intervals for seven days, repeatedly. The platelet count immediatelybefore,.days three and seven after the administration of cisplatin wasdetermined with a multi-functional automatic blood counter (SysmexE-7000). The results are shown in Table 1.

(2) Results

As shown in Table 1., a significant increase of platelet count wasobserved on and after day three of TCF-II administration. TCF-IImaintained a significantly higher platelet count on day seven than thatin control group of nine animals administered with the vehicle withoutTCF-II, which showed a marked decrease of platelet count.

Above results confirm the usefulness of TCF-II in the improvement ofsymptoms of various thrombocytopenia caused by the administration ofantitumor agent.

                  TABLE 1                                                         ______________________________________                                        Changes of platelet count in cisplatin administered rat.sup.a)                       Cisplatin administered rat                                                    TCF-II     Control   Healthy rat                                       ______________________________________                                        No. of animals                                                                         10           9         10                                            Before   108.8 ± 3.4                                                                             100.7 ± 3.9                                                                          110.5 ± 2.8                                administration                                                                Day three after                                                                        161.3 ± 5.2**                                                                           137.9 ± 6.0                                                                          131.6 ± 5.2                                administration                                                                Day seven after                                                                        32.8 ± 2.8**                                                                            19.9 ± 2.8                                                                           140.4 ± 2.9**                              administration                                                                ______________________________________                                         .sup.a :platelet count is expressed as mean ± S.D. and                     values with × 10.sup.3 /mm.sup.3.                                       **:indicates significant difference at a level of p<0.01                      from those in the control group.                                         

Usefulness for Industry

The agent for the treatment of abnormal blood coagulation of the presentinvention can be used for the treatment of disseminated intravascularcoagulation (DIC), and various symptoms accompanied with decreasedplatelet count.

We claim:
 1. A method for treating a blood coagulation disordercomprising disseminated intravascular coagulation (DIC) orthrombocytopenia, comprising administering to a subject afflicted withthe blood coagulation disorder a therapeutically effective amount ofTCF-II.
 2. The method of claim 1 wherein the blood coagulation disorderis accompanied by a decrease in platelet count resulting from decreasedproduction of platelets, accelerated platelet destruction, abnormaldistribution of platelets or abnormal platelet function.
 3. The methodof claim 1 wherein the mount of TCF-II administered comprises from about500 to about 3,000 μg/kg/day.
 4. The method of claim 1 wherein TCF-II isadministered by injection.
 5. The method of claim 1 wherein the TCF-IIis in admixture with a pharmaceutically acceptable carrier.
 6. Themethod of claim 1 wherein the TCF-II is recombinant.
 7. A method forincreasing platelet count in a subject afflicted with disseminatedintravascular coagulation (DIC) or thrombocytopenia comprisingadministering to the subject an effective platelet-increasing amount ofTCF-II.
 8. The method of claim 7 wherein the mount of TCF-IIadministered comprises from about 500 to about 3,000 μg/kg/day.
 9. Themethod of claim 7 wherein TCF-II is administered by injection.
 10. Themethod of claim 7 wherein the TCF-II is in admixture with apharmaceutically acceptable carrier.
 11. The method of claim 7 whereinthe TCF-II is recombinant.